The present invention relates to sterile containers for drying biological samples, and related methods. More particularly, the present invention relates to microcentrifuge tubes and methods of using the same.
It is often desired to dry biological samples in order to preserve their shelf life and activity. One drying technique is lyophilization, which is a commonly employed freeze-drying technique. Still other biological materials, such as long chain DNA molecules and cell components, are desired to be dried at a temperature above 0xc2x0 C., i.e., above freezing, in order to prevent their destruction by the forces of freezing. Inasmuch as the present invention is not limited to lyophilization, drying above and below the freezing point are discussed interchangeably.
Usually, after a lyophilization process is completed the freeze-dried compound is stored in a freezer, e.g., at xe2x88x9270xc2x0 C., although lyophilization can sometimes obviate the need for freezing all together. For example, according to the manufacturer""s product profile sheet, Endothelial Cell Growth Supplement (ECGS) is stable for at least 18 months when stored at 4xc2x0 C. in lyophilized form, but only one month when stored in a solubilized form at xe2x88x9220xc2x0 C.
Lyophilization of compounds is particularly useful when growing cells in a culture medium where the lyophilized compounds include peptides or growth factors. These compounds are generally provided in minute quantities due to their expense and/or potency, and they are usually extremely perishable. Lyophilization is observed to extend their shelf life.
Typically, lyophilization is carried out in a centrifugal apparatus, such as a Speed-Vac(copyright) centrifuge. The Speed-Vac(copyright) is placed in a vacuum chamber. The sample is placed in a microcentrifuge tube which is a small plastic tube (0.5, 1, or 2 mLs) typically tapered, conical or rounded, and closed at one end. Because the vacuum used in lyophilizing is extremely high (e.g., 50-500 millixc2x7Torr), some of the liquid in the microcentrifuge tube vaporizes immediately and forces out much of the remaining solution from the tube. By applying a centrifugal force, the liquid is pushed down to the bottom of the tube in an effort to prevent the liquid from jetting out when the liquid gassifies. After lyophilization is complete, the vacuum is turned off, thereby allowing the vacuum chamber and the interior volume of the tube to return to ambient pressure.
In order to use stored dried compounds, they must be dissolved (if not already stored in solution), then filtered-sterilized, which filters out all living cells, dust, and other unwanted materials. The volume of the solution at this stage is small, e.g., 1 ml. After filter-sterilization, the compounds are usually distributed in aliquots, e.g., of 50 xcexcl each, and unused aliquots are stored in a freezer. This avoids the necessity of repeatedly freezing and thawing the compounds, which shortens their shelf life.
Another lyophilization method entails leaving the microcentrifuge tube lid open during lyophilization. After the vacuum is terminated, the lid is then closed. This method produces an unsterile sample, which must be resterilized by filter-sterilization. However, in this method, that portion of the stored sample adsorbed to the filter is lost.
Still another method is to perform lyophilization in a sterile environment such as a clean room. However, this requires incurring the additional expense of maintaining a clean room environment.
Yet another method proposes sterile gas exchange through a membrane in an enclosed sterile environment, see, for example, U.S. Pat. No. 5,398,837 and a cell culture flask manufactured by Costar (catalog number 3056). However, neither of these methods is suitable for lyophilization using a centrifuge since the cell culture flasks cannot be centrifuged at high speeds. Moreover, the cell culture flasks provide a slow gas exchange between the outside environment and the cell culture being grown. Furthermore, the porosity of the membrane is such that it is permeable to gas but not to microbes, e.g., having diameters above about 0.22xcexc.
Accordingly, a need exists for a container for a material that can be subjected to high centrifugal forces, as during a drying procedure, but which permits sterile gas exchange between the interior of the container and the external environment. Such a container need only be capable of permitting drying while preventing microbial contamination, independent of centrifugation, for those applications not requiring centrifugation.
The present invention is for a method of drying a solid, liquid, or gaseous sample containing a vaporizable material, such as when drying a solid material of a liquid solvent for the solid material. Such a method comprises providing a container containing the sample, which container defines an opening with the opening sealed substantially by a filter element (means), such as a membrane. The filter element permits permeation of the vaporizable material, e.g., gas, solid, liquid, or combination thereof, while substantially preventing permeation of microbes into the container. The drying method further entails permitting at least a portion of the vaporizable material to permeate the filter means, thereby affording at least a partial drying of the sample without substantial microbial contamination thereof.
The present invention also is for a method of venting a sample to its surroundings. As used herein, xe2x80x9cventingxe2x80x9d refers to permitting the contents of a container to come into contact with a gas external of the container either by permitting a gas flow into the container from outside or by permitting volatile components within the container to pass the external environment. Such venting method entails providing a container having an opening sealed substantially with a filter means, which permits permeation of at least one gas and substantially prevents permeation of microbes. Preferably, the container is configured to withstand a high speed centrifugation of 50 or more times the force of gravity, and permits the gas to enter or exit the container by permeating through the filter means. Such method thereby affords venting of the sample in the container without substantially contaminating the sample with microbes.
A container assembly aspect of the invention comprises a container having a closed end and an open end, which defines an interior volume therein, with the container capable of withstanding centrifugation at about 50 or more times the force of gravity. The container assembly also comprises a cap having an open position and a closed position for sealing the open end of the container, which cap carries a microbe-impermeable filter means that permits gas flow into the interior volume from external the container and permits gas flow out from the interior volume.
Relatedly, a container assembly is also contemplated which comprises a container having a closed end and an open end that defines an interior volume therein, where the container is shaped to conform to the shape of a centrifuge rotor or bucket. The container assembly also comprises a cap having an open position and a closed position for sealing the open end, with the cap including a microbe-impermeable filter means as described hereinabove.
In a preferred embodiment an instant container is provided as a microcentrifuge tube. In another embodiment, the container can be a centrifuge bottle, which conforms either to a bucket that hooks onto a centrifuge rotor or conforms to a well provided in the rotor of the centrifuge. Such a centrifuge bottle usually has a capacity of 100 mL or greater, and has a flat bottom supported by the well or bucket into which it is placed.
Also contemplated is a method of making a container assembly, and associated cap, of the invention which entails providing a cap which defines an aperture therein, covering the entire aperture with a filter means that does not permit substantial permeation of materials having a diameter of at least about 0.2 microns, and securing the filter means to the cap with an adhesive, a cement, a welding, or a mechanical fastening.